The present invention concerns calibrators or calibration solutions which are based on a human serum matrix and which are used in a method for detecting cytokeratin, a process for producing them, a method for stabilizing cytokeratin in human serum and an immunological method for detecting cytokeratin in a sample.
Immunological detection methods have become very important in diagnostics. They are characterized by a high specificity and sensitivity and hence they are also very suitable for detecting low concentrations of analytes in biological fluids. Immunological detection methods are of major importance particularly in the fields of infectious diseases, fertility and thyroid diagnostics, for metabolic diseases and for diagnosing tumour diseases. At present carcino-embryonic antigen (CEA), alpha-fetoprotein (AFP), prostate-specific antigen (PSA) and cytokeratins (CK) are for example among the most significant tumour markers.
Human cytokeratins are building blocks of the intermediary filaments which are the major components of the cytoskeleton of epithelial cells. More than 19 cytokeratins are known of which cytokeratins 1 to 8 are referred to as basic cytokeratins and cytokeratins 9 to 19 are referred to as acidic cytokeratins. The cytokeratins can aggregate in the cell to form tetramers. A tetramer consists of two basic and two acidic cytokeratin molecules in each case. Linear aggregation of tetramers results in the formation of filaments. Intact cytokeratin molecules are water-insoluble as integral components of the intermediary filaments of epithelial cells. The complexity and composition of the cytokeratins differs in the various epithelial tissues i.e. epithelial cells have cytokeratin compositions that are typical for the respective tissue. The soluble fragments of cytokeratin 19 (CK 19) which are also referred to as CYFRA 21-1 are particularly relevant for tumour diagnostics. A concentration of CYFRA 21-1 that is increased in comparison to healthy persons indicates the presence of a tumour disease. Increased values have previously been found in the following tumours: bronchial carcinoma, ovarial cancer, cervix carcinoma, bladder carcinoma and in tumours of the head and neck. The main indication for CYFRA 21-1 is to monitor non-small cell bronchial carcinomas.
A method for detecting CK 19 by a sandwich ELISA technique is described for example in EP-A-0 460 190. In this method the body fluid sample to be examined is incubated with at least two receptors R1 and R2, R1 and R2 being monoclonal antibodies which each detect different epitopes of CK 19. One of the antibodies is labelled with biotin and the other carries a different label. The sandwich complex comprising R1, CK 19 and R2 binds to a solid phase coated with streptavidin. After separating the solid from the liquid phase, the label is measured in one of the two phases and preferably in the solid phase. The tumour marker CK 19 can be detected in the sample on the basis of the signal that is obtained.
When carrying out such a test it is important that the measured value that is obtained can be at least classified qualitatively as positive (tumour marker is present) or as negative (the tumour marker is not present). This applies especially to the classification of measurement data that are obtained by means of automated systems. It is often also desirable or necessary to quantify the concentrations of tumour marker. Hence the test system must be calibrated with reagents that contain a defined concentration of analyte before carrying out the measurements. These defined reagents are referred to in the following as calibrators. The terms calibration solution, calibration standard, standard solution or control are used synonymously for the term calibrator.
An important requirement for a calibrator is high stability. On the one hand it is necessary to ensure the accuracy of the test, an essentially 100% recovery of the analyte in the calibrator and a good signal to noise ratio. On the other hand a reliable reproducibility of the result of the determination must be guaranteed over a long time period. Hence calibrators must be insensitive to their environmental conditions over a time period of several weeks i.e. to temperature, direct solar radiation on the laboratory bench, pH value, buffer conditions etc. If the conditions are unfavourable there is a risk of hydrolysis, proteolysis or denaturation of the calibrator. The use of a calibrator that is no longer intact would lead to erroneous measurements.
Many tests are carried out in serum samples. In order to ensure comparability of the measurements, the calibrator should therefore also be based on a serum matrix. The more sensitive the measuring system, the larger the differences in measurement will become due to the jump in the matrix between sample and calibrator. It has been shown that cytokeratin is unstable in a serum matrix. When a calibrator containing cytokeratin is stored in a liquid state, the cytokeratin is destroyed and denatured and hence such a calibrator cannot be used for a long time period.
Hence in the past the serum matrix was replaced by an artificial matrix the composition of which mimics that of serum. The artificial matrix is generally based on a buffer to which various salts and proteins (for example bovine serum albumin) are added in order to simulate as closely as possible the natural human serum environment with regard to salt and protein concentration as well as pH value. Although this enabled the analyte cytokeratin to be stabilized in the calibrator, a disadvantage of this procedure is that comparability is not optimal especially with sensitive measuring systems since the underlying matrix of the samples is human serum. There is therefore a jump in the matrix. This can lead to measuring errors with samples in the low measuring range near to the cut-off value. In extreme cases this could lead to a disparity between the value determined for a positive cytokeratin signal using the calibrator based on an artificial matrix and the value determined for a human serum sample even when the cytokeratin analyte is present in each case at a comparable concentration.
Hence the object was to provide a calibrator for methods for detecting cytokeratin which is based on a serum matrix and preferably a human serum matrix and which is stable for a period of at least several weeks even under unfavourable ambient conditions.
The object is achieved by a calibrator whose essential components are serum, preferably human serum, cytokeratin and aprotinin. It surprisingly turned out that the addition of the protease inhibitor aprotinin can effectively stabilize cytokeratin. Consequently it is possible to provide a calibrator for cytokeratin detection methods which is manufactured on a natural serum matrix basis and in particular is based on a human serum matrix. This avoids a jump in the matrix when measuring the actual serum samples.
Furthermore it turned out that the calibrator according to the invention has a long shelf life and temperature stability. Thus the recovery rate for the cytokeratin analyte dissolved in the calibrator, preferably CK 19, is almost 100% in a sandwich immunoassay after stressing the lyophilised calibrator for three weeks at 30 to 40xc2x0 C. Thus the cytokeratin is not destroyed and is still immunologically recognized by the antibodies used in the test even after the time-temperature stress. The measured concentrations of cytokeratin in the various calibrators measured at the start of the stability test essentially correspond to the concentrations after the stress test. Hence the stability and precision of the calibrator according to the invention is comparable with the stability of a corresponding calibrator based on an artificial matrix. Even when the calibrator is stored at 2 to 8xc2x0 C. in a liquid or reconstituted form which corresponds to the usual storage temperature of reagents in a refrigerator, a cytokeratin recovery of almost 100% was found even after 10 weeks. This means that the calibrator according to the invention can still be used after long storage in a refrigerator without qualitative or quantitative impairment.
The protease inhibitor aprotinin is of major importance for the substantially improved stability of the calibrator according to the invention. Other protease inhibitors proved to be ineffective. Aprotinin is a commercially available polypeptide which is composed of 58 amino acids. It has an inhibitory effect on the coagulation factors XIIa, XIa and VIIIa as well as on plasmin and plasmin activators, as well as trypsin, chymotrypsin and kallikrein. surprisingly other known protease inhibitors have proven to be unsuitable for stabilizing the calibrator. Experiments with other substances such as detergents and salts did not result in the desired stabilizing effect.
Aprotinin is preferably used at a concentration of at least 10 mg/l in the calibrator. The maximum possible concentration is that which interferes with the test or at which turbidity starts due to the lack of solubility. Concentrations between 25 and 40 mg/l are particularly preferred.
The calibrator can either be prepared in a liquid form or as a lyophilisate. In order to prepare the lyophilisate, all liquid and solid components are firstly mixed together or dissolved and subsequently lyophilised. The lyophilisate is then finally used as a reconstituted solution i.e. it is dissolved again in liquid. Distilled water is usually used for the reconstitution i.e. to redissolve the lyophilisate since this does not add undesired ions to the calibrator and in particular does not change the salt concentrations. The amount of water depends on the desired cytokeratin concentration or on the desired fill volume.
The calibrator according to the invention can additionally also contain conventional substances known to a person skilled in the art such as salts or additional preservatives. For example N-methyliso-thiazolone and oxypyrion are preferably added at the usual concentrations of about 1 mg/l.
The calibrator according to the invention can preferably be stored in a lyophilized form at 4xc2x0 C. for several months without loss of quality. It can be stored for a period of up to 36 months at 4xc2x0 C. The calibrator can be stored in a reconstituted form for several months at 4xc2x0 C.
Cytokeratin-free human serum is preferably used as the serum. This can be obtained by affinity chromatographic processing or a serum is used which is free of cytokeratin from the beginning which has to be determined by appropriate screening methods.
The invention additionally concerns a process for producing the calibrator according to the invention. The calibrator is preferably produced by the following steps
a) mixing serum with aprotinin
b) filtering the solution
c) dissolving the cytokeratin in water
d) mixing the solution from step b) with the dissolved cytokeratin from step c)
e) lyophilizing the solutions from d)
f) dissolving the lyophilisate in water before use.
If necessary the pH value can be adjusted in step d) to pH 7 to 8, preferably 7.2.
A further subject matter of the invention is also a process for producing stabilized cytokeratin which is characterized in that the protease inhibitor aprotinin is added to the preferably purified cytokeratin. The stabilized cytokeratin produced in this manner is preferably used in a calibrator.
Another subject matter of the invention is the use of aprotinin to stabilize cytokeratin and in particular CK 19.
A subject matter of the invention is also a method for stabilizing cytokeratin in serum, preferably human serum which is characterized in that aprotinin, preferably at a concentration of at least 10 mg/l, is added to the serum.
A further subject matter of the invention is an immunological method for determining cytokeratin in a sample and in particular to determine CK 19. The method is characterized in that the signal determined for the sample is compared with the signal which is obtained with the aid of the calibrator according to the invention, the calibrator being measured using the same method as for the sample.
Such a method for determining cytokeratin and preferably for determining CK 19 and the calibrator is preferably carried out using the following steps:
a) reacting the sample with a first binding partner that is specific for cytokeratin and carries a group capable of binding to a solid phase which can be used to bind it to a solid phase,
b) reacting this solution with a further binding partner which carries a label
c) binding the immune complex that is formed to a solid phase, whereby the solid phase can already be present in step a)
d) separating the solid from the liquid phase
e) detecting the label in one of the two phases.
f) comparing the measured values for the calibrator with the value for the sample and quantification.
The method can also be carried out as a competitive test by methods known to a person skilled in the art.
The binding partners are preferably monoclonal or polyclonal antibodies or fragments thereof such as F(abxe2x80x2)2, Fabxe2x80x2 or Fab fragments which can specifically immunologically recognize and bind cytokeratin and in particular CK 19. The antibodies are produced by methods familiar to a person skilled in the art. Antibodies are also included which have been produced by modifying the antibodies for example by genetic engineering. The term antibody includes all aforementioned meanings for binding partners.
The first specific binding partner for cytokeratin can either be directly bound to the solid phase or the binding to the solid phase occurs indirectly via a specific binding system. The direct binding of this binding partner to the solid phase occurs according to methods known to a person skilled in the art. If the binding is indirect via a specific binding system, then the first binding partner is a conjugate comprising an antibody to cytokeratin and one reaction partner of a specific binding system. In this case a specific binding system is understood as two partners that can specifically react one another. The binding capability can be based on an immunological reaction or on another specific reaction. A combination of biotin and avidin or biotin and streptavidin is preferably used as the specific binding system. Other preferred combinations are biotin and antibiotin, hapten and anti-hapten, Fc fragment of an antibody and antibody to this Fc fragment or carbohydrate and lectin. One of the reaction partners of the specific binding pair is then a part of the conjugate.
The other reaction partner of the specific binding system for the first binding partner is present as a coating on the solid phase. The other reaction partner of the specific binding system can be bound to an insoluble carrier material by conventional methods known to a person skilled in the art. In this case a covalent as well as an adsorptive binding is suitable.
Suitable solid phases are test tubes or microtitre plates made of polystyrene or similar plastics whose inner surface is coated with a reaction partner of the specific binding system. Other suitable and particularly preferred solid phases are particulate substances such as latex particles, magnetic particles, molecular sieve materials, glass beads, plastic tubes etc. Porous layer-like carriers such as paper can also be used as carriers. Magnetic particles, so-called beads, are particularly preferably used and are in turn coated with the appropriate binding partner of the specific binding system described above. In order to carry out the detection reaction, these microparticles can then be separated from the liquid phase after completion of the test reaction for example by filtration, centrifugation or by a magnet in the case of magnetic particles.
The specific binding reactions between the antibodies to cytokeratin and cytokeratin can be detected in various ways. Usually one binding partner of the specific binding reaction is labelled. Common labels are chromogens, fluorophores, substances capable of chemiluminescence or electrochemiluminescence, radioisotopes, haptens, enzyme labels or substances which can in turn form a specific binding pair such as biotin/streptavidin.
All biological fluids familiar to a person skilled in the art can be used as samples to carry out the method for the detection of cytokeratin. Body fluids are preferably used as the sample such as whole blood, blood serum, blood plasma, urine or saliva, particularly preferably blood serum.